PROTOCOL PCR HOTSTART
1. prepare product sample ekstraksi, then contents form pcr. count sample and make mastermix for sample total augmentings control negative n add 1 to eror pipetting. our for example has sample 5, add control negative 1 and eror 1 so we make mastermix to 7 tubes. if has which are positive result, we also can add control positive. add 1 eror to every multiple 10.2. prepare all reagen (h2o, buffer pcr red, dntps, mgcl2, and primary forward and revers.
2. take 2 tubes strip complete with enclosing it. give initial label, date, and primary. then write number 1 until 8 in tube neck strip. tube strip the other one let without label. prepare 2 tubes eppendrof 0,5 ml and give label mm1 and mm2.4. now, we liquefy all reagen (usually kept at frezer, certain it frozen, so it necessary liquefy)ed with hold tight and ascertain all melt finely. shakes to ascertain there is no component endap mendasar tube.
3. before memipet reagen, always mix beforehand by using pipette.
4. take h2o appropriate count that we can, and input to tube mm1 and also take to mm2 (volume appropriate protocol)
5. then take also to buffer mm1 and mm2. then enclosing mm2 and now we are working with mm2.8. take dntps, mgcl2 primary 1 and 2, then mix by using pipette.
6. take 14 µl mm1 and input has intoed every tube hole strip that we have given label.
7. input 1 µl dna result ekstraksi into tube strip yan full mm1 while at mix.
8. ascertain in tube there is no bubble. if necessary at spin.
9. furthermore open tube casing strip containing mm1 and dna, and enclosing only in hole 1 and 8.13. now we are working with mm2. and we take enzyme taq red, we are msu into tube mm2 and we are mix.
10. 14. for into every tube strip that still empty and not melabel a while ago as much as 11 µl mm2.15. furthermore prepare multi chanel pipette, setting in volume 11 µl. and tips.
11. lets go to thermocycler…. nasu tube strip containing mm1 and dna into thermocycler, enclosing and running engine with program hotstart. wait up to engine temperature demoes temperature 80. press pause. open casing strip tube.
12. reopen engine, take mm2 as much as 11 µl with multichannel pipette, then input in tube strip that stay in engine, mix with pipette, then enclosing return tube and also engine with crush kembalu run. (try to do swiftly and correct. longer the work, enzyme less work)
13. wait up to process finished. and neat dehhhhh.